Supplementary Figure 3: SCRIB interacts directly with p22phox, which is required for PMA-induced generation of ROS.

(a) Schematic showing the full length SCRIB protein with Leucine Rich Repeats (LRR) and PDZ domains and the 6xHIS-tagged individual SCRIB PDZ domains or GST-tagged p22phox C-terminal tail (GST-p22tail). (b) ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectrum of ¹⁵N- labeled ScrPDZ4 (0.2 mM) in the absence (black) and the presence of varying concentration ratio of p22phox tail peptide/SCRIBPDZ4: 1.5 (red), 3.0 (green), 4.5 (blue), 8.9 (yellow), 17.8 (magenta) and 26.7-fold (cyan), are shown. Assignments were transferred from human SCRIB PDZ4 domain (BMRB number 10103) and further confirmed by 3D NMR experiments. Residues exhibiting large chemical shift (> 0.2 normalized chemical shift change in Supplementary Fig. 3c) are indicated. An asterisk (∗) indicates the peak from N-Acetylglycine-¹⁵N. Repeated 4 times and the representative experiment was shown as a figure. (c) NMR characterization of the interaction between p22phox tail and His-SCRIBPDZ4. The chemical shift change of each SCRIBPDZ4 residue is normalized to the observed change for G1112 in the presence of the p22phox tail peptide (26.7-fold). Blank regions indicate residues for which the value cannot be determined because of overlapped or unassigned peaks. Residues exhibiting large chemical shifts (> 0.2 normalized chemical shift change) are labeled. (d) SCRIBPDZ4 residues affected by the addition of p22phox tail peptide are mapped and indicated on the apo SCRIBPDZ4 structure (that is residues 8–105 from PDB ID 1UJU). (e) Electrostatic potential surface representation of SCRIBPDZ4 at neutral pH. Basic and acidic potentials are shown in blue (+5 kT/e) and red (− 5 kT/e), respectively. Basic residues near p22phox binding pocket are indicated. The electrostatic surface potential was generated using the APBS tools in Pymol, with partial charges determined by the PDB2PQR server (http://nbcr-222.ucsd.edu/pdb2pqr_1.8). (f) Conservation of SCRIBPDZ4 residues. Percent conservation of each residue position in human SCRIBPDZ4 (Residues within R1099-C1189) compared to the alignment of mouse, rat, zebrafish and fly SCRIB PDZ4 protein sequences. The residues affected by p22phox tail binding are shown. (g) ¹H-¹⁵N HSQC spectrum of ¹⁵N-labeled ScrPDZ4 (0.2 mM) in the absence (black) and the presence (blue) of the full p22phox cytoplasmic region (that is residues 131–195, 5.0-fold excess). Residues exhibiting large chemical shift in Supplementary Fig. 2 are indicated. Repeated twice and the representative experiment was shown as a figure. (h) ¹H-¹⁵N HSQC spectrum of ¹⁵N-labeled SCRIBPDZ4 (0.2 mM) in the absence (black) and the presence (green) of the p22phox cytoplasmic region with a C-terminal tail truncation (that is residues 131–185, 5.0- fold excess). An asterisk (∗) indicates peaks from N-Acetylglicine-¹⁵N. Repeated twice and the representative experiment was shown as a figure. HSQC spectra of wild-type i and G1112A/R1178A double mutant (j) ¹⁵N-labeled ScrPDZ4 (0.1 mM) in the absence (black) and the presence (red) of p22phox tail peptide (32.4-fold) are shown. Repeated twice and the representative experiment was shown as a figure. k CD spectra of the wild type (WT) and G1112A/R1178A double mutant forms of Scrib-PDZ4. (l) Comparison of response curves of WT and G1112A/R1178A variants of ScribPDZ4. Response curve was obtained with 20 μM ScribPDZ4 proteins.